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Technical Brief 

T-Cell and B-Cell Clonality Using BIOMED-2 PCR Primers 

Background Information 

An assessment of T-cell or B-cell clonality is an important 

part of the evaluation of suspected lymphoproliferative 

disorders. Historically, Southern blot studies for T-cell 

receptor beta chain (TCRB) rearrangements have been 

considered to represent the gold standard for T-cell clonality 

evaluation while immunoglobulin heavy chain (IGH) and/or 

immunoglobulin kappa light chain (IGK) rearrangements 

have served this purpose for B-cell clonality. Southern blot 

studies, however, are labor-intensive and time-consuming for 

the laboratory and often are impractical for routine practice 

as they require fresh or frozen tissue and cannot be performed 

on formalin-fixed, paraffin-embedded (FFPE) material. 

PCR assays for T-cell receptor gamma chain (TCRG) and/or 

TCRB rearrangements and IGH or IGK rearrangements offer 

the ability to assess clonality from standard FFPE, but until 

recently, PCR studies have been limited by a higher false 

negative rate compared to Southern blot studies. 

The BIOMED-2 multinational collaborative study developed 

and standardized multiplexed PCR primers that are capable 

of detecting clonal lymphocyte populations with a sensitivity 

that approaches that of Southern blot (1). Cleveland Clinic 

Laboratories now offers T-cell clonality and B-cell clonality 

assays using BIOMED-2 PCR primers. 

The T-cell clonality assay employs primers for both TCRB and 

TCRG, a combination that has been shown to detect clonality 

in essentially 100% of T-cell prolymphocytic leukemias, T-cell 

large granular lymphocyte disorders, and peripheral T-cell 

lymphomas, unspecified, with somewhat lower rates reported 

in angioimmunoblastic T-cell lymphomas and anaplastic 

large cell lymphoma (2). Assays for only TCRB or only TCRG 

rearrangements may also be ordered, if desired. 

The B-cell clonality assay employs primers for both IGH and 

IGK. This combination of IGH and IGK primers has been 

shown to detect approximately 98% of B-cell clonal populations 

compared to Southern blot (3). Assays for only IGH or 

only IGK rearrangements may also be ordered, if desired. 

Clinical Indications 

These assays are designed for detection of clonal T-cell or 

B-cell populations in suspected lymphoproliferative disorders 

using fresh, frozen, or FFPE tissue. 

Interpretation 

Results are reported as: 

• Positive for a clonal population, 

• Negative for a clonal population, or 

• Indeterminate. 

Limitations of the Assays 

1. Results of clonality studies must be interpreted in the 

context of the clinical and histologic findings. Clonality 

is not equivalent to malignancy as physiologic clonal 

populations may be detected in some reactive conditions. 

2. For optimal detection of T-cell clonality, the use of both 

TCRB and TCRG primers is recommended. 

3. For optimal detection of B-cell clonality, the use of both 

IGH and IGK primers is recommended. 

4. The detection of TCRB and/or TCRG rearrangements (for 

T-cell clonality) or IGH and/or IGK rearrangements (for 

B-cell clonality) cannot be used for lineage assignment, 

as some T- or B-cell lymphoproliferative disorders or 

acute myeloid leukemias may have detectable clonal 

rearrangements with these primers. False positive 

“pseudoclonal” results may sometimes be detected when 

few T- or B-cells are present in the tissue analyzed. Rare 

clonal T-cell or B-cell populations may not be detected 

using these primers. 

01.31.11

Methodology 

PCR is performed utilizing the standardized BIOMED-2 

protocol (1). 

PCR products are analyzed using capillary electrophoresis. 

For T-cell clonality: Rearrangements of the TCRB locus are 

assessed using three sets of labeled multiplexed PCR primers. 

The first two tubes consist of forward primers targeting V 

variable segments and reverse primers targeting the J joining 

region. The third TCRB tube detects incomplete rearrangements 

using forward primers to the D diversity segments and 

reverse primers targeting the J joining regions. Rearrangements 

of the TCRG locus are assessed using two sets of multiplexed 

labeled PCR primers, each with forward primers for the V 

variable regions and reverse primers targeting the J joining 

region. 

For B-cell clonality: Rearrangements of the IGH locus are 

assessed using three sets of labeled multiplexed PCR primers. 

These tubes consist of forward primers targeting the IGH 

variable framework 1, framework 2, or framework 3 region 

with common reverse primers to the JH joining region. 

Rearrangements of the IGK locus are assessed using two 

sets of multiplexed labeled PCR primers. The first tube uses 

forward primers for the kappa variable regions and a reverse 

primer targeting the J joining region. The second tube uses 

forward primers to the kappa variable regions and an intron 

primer together with a reverse primer targeting the kappa 

locus deleting element. 

References 

1. JJM VanDongen, AW Langerak, M Bruggermann et al. 

Design and standardization of PCR primers and protocols 

for detection of clonal immunoglobulin and T-cell receptor 

gene recombinations in suspect lymphoproliferations: 

Report of the BIOMED-2 Concerted Action BMH4-CT98- 

3936. Leukemia. 2003;17:2257-2317. 

2. M Bruggermann, H White, P Gaulard et al. Powerful 

strategy for polymerase chain reaction-based clonality 

assessment in T-cell malignancies: Report of the 

BIOMED-2 Concerted Action BHM4-CT98-3936. 

Leukemia. 2007;21:215-221. 

3. PAS Evans, Ch Pott, PJTA Groenen et al. Significantly 

improved PCR-based clonality testing in B-cell malignancies 

by use of multiple immunoglobulin gene targets. 

Report of the BIOMED-2 Concerted Action BHM4-CT- 

98-3936. Leukemia. 2007;21:207-214.

Test Overview 

Test Name 

T-Cell Clonality Using BIOMED-2 

PCR Primers 

T-Cell Receptor Beta BIOMED-2 

PCR 

TCR-G (PCR) 

Reference Range 

Negative for clonal rearrangement 



Specimen Requirements 

External Specimen Requirements: 

Testing Volume/Size: 10 mm 2 

Type: Tissue, frozen 

Tube/Container: Clean container 

Note: Frozen tissue should be 

delivered to Surgical Pathology for 

accessioning and cutting. 


Type: Tissue, paraffin-embedded 


Note: Paraffin-embedded tissue 

should be delivered to Surgical 

Pathology for accessioning and 

cutting. 

Alternate Specimen Requirements: 

Testing Volume/Size: 2 mL 

Type: Bone marrow 

Tube/Container: EDTA (Lavender) 

Transport Temperature: Refrigerated 


Type: Fluid, body 



Note: Fluid must contain at least 

3 million cells. 


Type: Whole blood 







Transport Temperature: Frozen 


should be delivered to Anatomic 


cutting. 







cutting. 
















Testing Volume/Size: Other 

Type: Extracted DNA 



Note: Volume/Size: 6µg 






Note: Send specimen at -70°C on 

dry ice. 




Transport Temperature: Ambient 
















Test Ordering Information 

Clearly indicate specimen source 

on sample label 





Billing Code 87903 

CPT Code 83891; 83898; 83900; 

83901(x7); 83909(x5); 83912 

87965 

83891; 83898; 83900; 

83901(x3); 83909(x3); 83912 

81402 

83891; 83900; 83901(x4); 

83909(x2); 83912

Cleveland Clinic Laboratories 

9500 Euclid Avenue, L15, Cleveland, Ohio 44195 

800.628.6816 | clevelandcliniclabs.com 

Test Overview 

Test Name 

B-Cell Clonality Using BIOMED-2 

PCR Primers 

Immunoglobulin Heavy Chain 

Using BIOMED-2 PCR Primers 

Immunoglobulin Kappa Chain 

Using BIOMED-2 PCR Primers 

Reference Range 




Specimen Requirements 



Type: Tissue 


Note: May submit fresh, frozen or 

paraffin-embedded tissue 


















Type: Tissue 



Note: Frozen and fresh tissue 



cutting. Paraffin-embedded tissue 



cutting. 







Type: Blood 







Note: Volume/Size: 6 µg 



Type: Tissue 



Note: Frozen and fresh tissue 



cutting. Paraffin-embedded tissue 



cutting. 







Type: Blood 







Note: Volume/Size: 6 µg 

Test Ordering Information 







Billing Code 87904 

CPT Code 83891; 83898(x3); 83900; 

83901(x2); 83909(x5); 83912 

87960 

83891; 83898(x3); 83909(x3); 

83912 

87954 

83891; 83900; 83901(x4); 

83909(x2); 83912 

Technical Information Contact: 

Kelly Lyon, MT 

216.444.8283 

palinck@ccf.org 

Scientific Information Contact: 

James R. Cook, MD, PhD 

216.444.4435 

cookj2@ccf.org 

201101.025

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